1. Field of the Invention
This invention is directed to methods for synthesizing very large collections of diverse tetramic acids (i.e., 2,4-pyrrolidinediones) and derivatives thereof, including tetronic acids, thiotetronic acids, pentamic acids, pentonic acids, and thiopentonic acids. This invention is further directed to methods for identifying and isolating tetramic acid compounds and derivatives thereof with useful and diverse activities from such collections.
2. State of the Art
Compounds having biological activity can be identified by screening diverse collections of compounds (i.e., libraries of compounds) produced through either molecular biological or synthetic chemical techniques. Such screening methods include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having biological activity or where the library comprises a plurality of compounds synthesized at specific locations on the surface of a solid substrate wherein a receptor is appropriately labeled to identify binding to the compound, e.g., fluorescent or radioactive labels. Correlation of the labeled receptor bound to the substrate with its location on the substrate identifies the binding compound.
Central to these methods is the screening of a multiplicity of compounds in the library and the ability to identify the structures of the compounds which have a requisite biological activity. Preferably, in order to facilitate synthesis and identification, the compounds in the library are typically formed on solid supports wherein the compound is covalently attached to the support via a cleavable or non-cleavable linking arm. In this regard, libraries of diverse compounds are prepared and then screened to identify "lead compounds" having good binding affinity to the receptor.
Pharmaceutical drug discovery relies heavily on studies of structure-activity relationships wherein the structure of "lead compounds" is typically altered to determine the effect of the alteration on activity. Alteration of the structure of the lead compounds permits evaluation of the effect of the structural alteration on activity. Thus libraries of compounds derived from a lead compound can be created by including derivatives of the lead compound and repeating the screening procedures.
Ideally, the compounds are synthesized in situ on the solid support so that the support can be tagged to identify the synthetic steps employed and/or the derivative incorporated onto the support. However, relatively simple synthetic methods to produce a diverse collection of such derivatives on the supports are often not available.
One particular class of compounds which would be useful for inclusion in screening libraries are tetramic acids compounds. 3-Acyl-2,4-pyrrolidinediones (acyltetramic acids) are a class of natural products that have attracted significant attention over the years as a result of their biological activities and the synthetic challenges presented by their complex structures. Representative examples of this structural class include streptolydigin, tirandamycin A, BU2313A and BU2313B which are natural products extracted from fungi that exhibit potent antibacterial activities. Streptolydigin and tirandamycin exhibit potent activity against Gram-positive organisms by inhibiting terminal DNA transferase and bacterial RNA polymerase enzymes. BU2313A and BU2313B are broad-spectrum antibiotics effective against both Gram-positive and Gram-negative anaerobic bacteria as well as some aerobic bacteria including Streptococci. Tirandamycin and the BU2313 compounds have exhibited efficacy in mouse protection tests, and tirandamycin in a mouse abscess model for Bacteroides fragilis. ##STR1##
Although the complicated polycyclic side chains of the above antibiotics may be required for inhibition of RNA polymerase and DNA transferase, the 3-acyl-2,4-pyrrolidinedione unit itself offers significant potential as a drug scaffold. With a pKa of 5, the ability to chelate metals, and the two ketone units, acyltetramic acids possess a number of potential binding modes with biological targets.
Tetramic acids and derivatives thereof are also valuable chiral starting materials for the synthesis of other natural and unnatural products.
A variety of solution phase techniques have been developed to prepare tetramic acids. For example, it is known to condense activated N-protected alpha-amino acids with Meldrum's acid to yield the corresponding 1-hydroxyalkylidene Meldrum's acid. Upon heating in solution acetone and CO.sub.2 are eliminated to form N-protected 5-substituted tetramic acids. See Jouin et al. (1987) J. Chem. Soc. Perkin Trans. I 1177. Alternatively, alpha-amino acid esters can be reacted with malonic acid ester chlorides to yield the corresponding N-(alkoxycarbonylacetyl)-alpha amino acid esters. The latter are cyclized to the 3-alkoxycarbonyl tetramic acids. See Mulholland et al. (1972) J. Chem. Soc. Perkin Trans. I 2121. See, also, U.S. Pat. No. 5,008,402, U.S. Pat. No. 5,534,540, U.S. Pat. No. 5,468,774, U.S. Pat. No. 4,996,227, and U.S. Pat. No. 5,498,732.
However, the incorporation of a multiplicity of tetramic acid derivatives on solid supports is not previously known. The ability to synthesize a multiplicity of tetramic acid derivatives on a solid support or on different solid supports would enhance the structural variation of a library and provide important structure-activity information.